Nutrients or resin? – The relationship between resin and food foraging in stingless bees

Abstract Stingless bees are important pollinators in tropical forests. Yet, we know little about their foraging behavior (e.g., their nutritional requirements or their floral sources visited for resource collection). Many stingless bees not only depend vitally on pollen and nectar for food but also on resin for nest building and/or defense. However, it is unclear whether the large effort devoted to collecting resin as a non‐food resource by certain stingless bees affects their foraging behavior. Therefore, in this study, we analyzed differences in foraging patterns (i.e., foraging activity, proportion of collected resources, and specialization in plants visited) and resource nutritional composition (i.e., sucrose amount in nectar and amino acids in pollen) of seven different stingless bee species (eleven wild colonies) in north‐western Ecuador with a particular focus on the role of resin collection. We found that species with a high resin intake tended to be more active than species with a low resin intake. The foragers per minute invested for pollen collection were similar across all species. Sucrose intake per minute differed between some species but was not affected by increased resin intake. Interestingly, high and low resin collectors partly differed in the plants visited for pollen collection. Pollen amino acid profiles largely, but not completely, overlapped between the two resin collection groups. Our findings show that the foraging patterns and plant choices of stingless bees may vary depending on their resin intake, highlighting the need for more research focusing on resin collection and use by stingless bees.

Besides nectar and pollen, stingless bees also collect other resources (Roubik, 1989).For many stingless bee species, plant resins play a vital role (e.g., Leonhardt, 2017).They are not only waterproof and sticky, but also often fungicidal, bactericidal, and bacteriostatic, making them the ideal material for construction and defense (Armbruster, 1984;Chui et al., 2022;Greco et al., 2010;Leonhardt & Blüthgen, 2009;Roubik, 2006).Moreover, resin compounds enrich the chemical diversity of the cuticular profile of some species, which can modulate their aggression toward other species (Leonhardt, 2017).Species differ strongly in the amount and proportion of resin foragers (e.g., Layek & Karmakar, 2018;Leonhardt et al., 2007Leonhardt et al., , 2014;;Leonhardt & Blüthgen, 2009;Wallace & Lee, 2010).Levels of resin use vary depending on predator pressure (Leonhardt & Blüthgen, 2009), colony developmental stage, and species characteristics, such as nest building (i.e., the amount of resin mixed with wax to build nest structures) and defense strategies (e.g., coating nest entrance with sticky resin layer or directly applying resin to predators, physical defense, such as biting, or hiding and closing the nest entrance) (Shanahan & Spivak, 2021) (see Table 1 for our study species).However, the factors influencing variation in the amount of resin collection and use as well as its effect on colony dynamics are still unclear.For example, for those species that use large amounts of resin for nest construction or defense, there could be a tradeoff between collecting resin and collecting food resources as pollen cannot be mixed with resin (Armbruster, 1984).In fact, a large foraging effort assigned to resin as a non-food resource may require some sort of compensation, for example, by increasing overall activity or collecting food resources from different plant species with different nutritional quality (e.g., high protein or sucrose content).
In this study, we explored differences in foraging patterns (returning foragers/minute, proportion of collected resources, and specialization in pollen sources, that is, plants and plant life forms visited) sino también de resinas para la construcción de su nido y/o defensa.Sin embargo, no está claro si el gran esfuerzo dedicado a la recolección de resina como recurso no alimentario de ciertas abejas sin aguijón afecta su comportamiento de forrajeo.
TA B L E 1 Colony name (ID) of stingless bee species found in the natural reserves Río Canandé and Tesoro Escondido.

Canandé
Apply resin to predators (Wittmann, 1985) Solitary.Poor recruiters (Aguilar et al., 2005) Resin around nest entrance (C.Rasmussen pers.obs.) Yes, high (57.81± 18.18) Note: The table includes information on: (a) the colony and species name, (b) the reserves where the colonies were located, (c) defense and foraging strategy of each species, (d) whether the nest entry is made solely out of wax or whether resin is used (with respective references), (e) whether they incorporate resin compounds into their cuticular profile (cp), (f) resin foraging behavior (mean percentage (%) ± SD of foragers returning with resin, obtained with the data of the 5 days observation), and (g) resin collection classification: low (≤20% resin foragers and absence of resin-derived compounds in their cuticular profiles) or high resin collectors (>20% resin foragers and presence of resin-derived compounds in their cuticular profiles).
and resource nutritional composition (sucrose amount in nectar and amino acids profiles of pollen) of 11 stingless bee nests representing seven species, with particular focus on the role of resin collection.
We expected differences in foraging activity among species as documented by Leonhardt et al. (2007).Additionally, we anticipated that bees with higher foraging activity (measured as foragers per minute) would also exhibit higher sucrose intake per minute, as observed for Tetragonula carbonaria, (Leonhardt et al., 2014).We also expected that stingless bees show little specialization in pollen collection, but have preferences for specific plant life forms, as stingless bees are broadly polylectic, but forage preferentially in either the understory or canopy (Nagamitsu et al., 1999).Finally, we also expected that they collect pollen with similar amino acid profiles as seen for bumblebees (Kriesell et al., 2017).
To understand the effect of resin collection on the bees' foraging behavior, we classified our study species as low and high resin collectors (see Section 2, Table 1).We hypothesized that high resin collectors show an overall higher foraging activity and collect pollen of higher protein content and nectar of higher sucrose content compared to low resin collectors to compensate for the workforce (i.e., foragers/minute coming back with one specific resource) invested in the allocation of a non-food source.Finally, we investigated whether the high resin collectors collected pollen from plants that also produce resin (in different foraging trips), as this might save the colony search efforts.

| Bee sampling and foraging observations
Eleven wild stingless bee colonies were located by walking through the forest (Figure 1, Table 1).For each colony, we sampled specimen bees for identification by CR and DNA barcoding using the BOLD Identification System (Ratnasingham & Hebert, 2007)  We classified our studied species into two groups (low and high resin collectors) based on their resin collection behavior (species with ≤20% resin foragers or with >20%, respectively, Table 1), and on the absence/presence (respectively) of resin-derived compounds in their cuticular profiles (using information from Leonhardt et al. (2013), Drescher et al. (2019), and analyzing their cuticular profiles (Figure S1, Appendix S1)).
Each nest was visited between February and April 2019 (rainy season) on five non-consecutive and non-rainy days to randomize F I G U R E 2 Employed methods for bee sampling and foraging observations.At the beginning of the observation period, returning foragers were recorded for 3 min to measure foraging activity.Afterwards, 20 different returning foragers (10 in the case of Plebia sp., as there were only very few active foragers) were captured and their corbicula and nectar loads were recorded.We used a 5 μL microcapillary tube to measure the sugar concentrations of nectar loads by placing the tube near the forager's mouth and carefully squeezing its abdomen.The volume of the regurgitated nectar was quantified, and its concentration (in percentage) was measured using hand-held refractometers.
The pollen loads carried on each leg were collected in different vials, frozen, and stored for amino acid and DNA metabarcoding analyses.Finally, at the end of the observation period, all returning foragers were again recorded for 3 min.Bee photo by Philipp Hoenle.
data collection across the entire study period and rule out effects of climatic variation over the day.Note that we did not collect environmental data.The observations were done at specific points of time during the day (between 8 am and 5 pm) and lasted around 2 h per colony with points of times randomized over subsequent visits.
Occasionally, in Tesoro Escondido reserve, nests were visited on the consecutive day, but at a different time than the previous day.
Each day, 20 different returning foragers (10 in the case of Plebia sp., due to few foragers) were captured using an insect net, and their corbicula and nectar load were visually checked (Figure 2).Loads of each bee were categorized into no load (nl), only nectar (including all fluids regurgitated from the crop) (n), only pollen (p), only resin (including all indistinguishable sticky substances) (r), pollen and nectar (pn), resin and nectar (rn), or resin and pollen (rp).In doing so, we could determine how much effort was allocated to each resource.
Captured bees were kept alive in plastic tubes, with holes in the lid to ensure ventilation, and released at the end of each observation period to avoid recapturing the same individual.
Each day, we video-recorded (camera Canon SX540 HS) the number of returning foragers for 3 min before and after capture (Figure 2).The average number of returning foragers/minute (i.e., activity, A) was calculated for each day and colony.To account for probable differences in colony size, daily activity was normalized (A1) using the maximum number of returning foragers/minute for each colony (max; A1 = A/max).We then calculated the number of foragers/minute with the load categories mentioned above (Fm (nl,n,p,r,pn,rn,rp) ) following Leonhardt et al. (2014): where P (nl,n,p,r,pn,rn,rp) is the proportion of foragers that carried a particular resource.
All captured bees were carefully squeezed to provoke regurgitation.The volume of regurgitated nectar was quantified in 5 μL microcapillary tubes, and its concentration (c, in percentage) was measured to the nearest 0.5 g/g sucrose equivalent by hand-held refractometers (Eclipse 45-81 (0-50 °Brix) and Eclipse 45-82 (45-80 °Brix), Bellingham + Stanley) (Figure 2).The sugar percentage was converted into x (μg/μL) following Leonhardt et al. (2014): The sucrose amount (ms, in mg) was obtained by multiplying x by the nectar volume and transformed into milligrams.We finally calculated the sugar intake (mg/min) (S in ) as follows: where P (Pn+Ppn+Prn) is the sum of the proportion of all foragers returning with nectar.
The pollen loads carried on each leg were collected in different vials, frozen, and stored for chemical and DNA metabarcoding analyses (Figure 2).For DNA metabarcoding, pollen pellets were sequenced individually.For the chemical analysis, pollen pellets from all pollen foragers collected for one colony at 1 day were pooled to obtain sufficient material for the analysis.Additional pollen foragers were captured for colonies BN1, BN2, BN3, BN4, and BN8.

| Pollen metabarcoding and plant assessment
We applied next-generation sequencing pollen DNA metabarcoding to construct bee-plant interaction networks (Bell et al., 2016(Bell et al., , 2017;;Keller et al., 2015;Pornon et al., 2017).DNA metabarcoding of the ITS2 plant region was performed following Sickel et al. (2015) with hands-on details provided in Campos et al. (2021), Ankenbrand et al. (2015), Edgar and Flyvbjerg (2015), Edgar (2016aEdgar ( , 2016b)), Afterwards, we also determined the life forms (i.e., epiphyte, herb, liana, shrub, tree, and tree or shrub) of the taxa accounting for more than 10% of reads per sample (after the 1% filter) and if they are known to produce resin based on taxonomic expertise of PL, Gentry (1996), the Global Biodiversity Information Facility (GBIF) (GBIF.org,2020), and the Catalog of the Vascular Plants of Ecuador (internet version) (Missouri Botanical Garden, 1999).For resin production, additional literature was consulted (Table S1).Note we could not identify the life form of some of the plants, which we then classified as "non determined".

| Analysis of pollen amino acids
Pollen protein-bound amino acids were analyzed by ion-exchange liquid chromatography (IEC, Biochrom 20 Plus Amino Acid Analyzer) following Leonhardt and Blüthgen (2012) (Appendix S1).We calculated the proportion of each amino acid and the total concentration of pollen amino acids (μg/mg of pollen, wet weight) in each sample.

| Statistical analysis
All analyses were performed with the software R version 4.2.1 (R Core Team, 2022).For all linear, linear mixed-effect models (lme4 Note: It is also indicated whether the data were transformed, and the tests used to obtain the p-values of the models.The functions and the R-packages (package citation is provided, when necessary) used to implement those tests are also given.Model's abbreviations: lm (linear models), glm (generalized linear models), lmm (linear mixed-effect models), glmm (generalized linear mixed models), and glmmTMB (generalized linear mixed models using template model builder).

| Differences in foraging patterns, resource intake, and nectar collection
We tested for differences in (a) foraging activity (A1) (the maximum values, i.e., A1 = 1, were excluded), and (b, c) proportion of returning foragers and foragers/minute with no load, only nectar, only pollen, only resin, pollen-nectar (T.angustula was excluded from this category as no forager came with these two resources together), resin-nectar (N.tristella and Plebeia sp. were excluded here for the same reason), and total pollen (P p + P pn + P rp /Fm p + Fm pn + Fm rp ) between species.The category resin-pollen was excluded as very few foragers across species and colonies returned with both resources simultaneously.Lastly, we analyzed differences in (d) nectar sucrose amount and intake/minute, and (e) pollen amino acid content (see Table 2 for the implemented statistical models).
To obtain valid models in some cases, we used glmmTMB with a log-linked hurdle-gamma distribution (zi Gamma) (Table 2), which overcomes the restriction of the classical gamma distribution that does not allow zero as a response (Brooks et al., 2017).Additionally, in two models "species" were used as dispersion parameters to account for heteroskedasticity (  S2), indicating that intraspecific differences and nest location did not crucially affect model results and could, therefore, be excluded (Figure S2 shows an example of a similar percentage of foragers for each resource for different colonies of the same species).

| Differences in plant species visited and in pollen amino acid profiles
We used bee-pollen-based interaction networks to depict pollen sources used by the different bee species (Bosch et al., 2009).The network analysis was made using RRAs (following Peters et al., 2022).We calculated the quantitative network-level specialization index, H 2 ′, and the species-level specialization index, d′ (Blüthgen et al., 2006) (bipartite R-package; Dormann et al., 2008).H 2 ′ and d′ range from 0 (in the case of H 2 ′: the different bee species visit similar plants for pollen collection, and in the case of d′: the specific species' pollen hosts overlap with other species) to 1 (in the case of H 2 ′: the different bees species visit different plants for pollen collection, and in the case of d′: the specific species' pollen hosts hardly overlap with other species).We used the null-model approach to see if our obtained H 2 ′ was significantly different from random networks (details in Appendix S1: Statistical analysis).For visualization, only the plant taxa accounting for more than 10% of reads were presented in the network.
Additionally, we analyzed differences in (a) the proportion of herbs, shrubs, and trees (life forms visited by almost all bee species) from which pollen was harvested, (b) the total concentration of pollen amino acids (Table 2), and (c) the proportional pollen amino acid profiles.For the latest, we conducted a classification analysis based on Breiman's random forest algorithm using species as the class predictor (package randomForest (Liaw & Wiener, 2002), 100,000 trees).With this analysis, we obtained the out-of-bag (OOB) estimate of error rate (which indicates the percentage of points that were misclassified in the training set) and the class errors for each species (which indicates grouping accuracy according to bee species: 0 indicates that all values were correctly classified and 1 that values could not be correctly classified).

Relationship between resin collection and general foraging patterns, resource intake, and nectar collection
To explore if intensive resin collection by high resin collectors might be compensated through increasing activity, we tested whether low and high resin collectors differed in (a) foraging activity and/or (b) the number of returning foragers/minute with each resource (see above).To assess if compensation might occur via intake of higher quality resources, we tested for differences in (c) sucrose amount and (d) sucrose intake/minute.For this, we used mixed-effect models with species as a random factor (Table 2).
Additionally, for each species, we tested if the number of resin foragers/minute was correlated with the number of only pollen and only nectar foragers/minute using Spearman correlation tests (cor. test function, stats package).
To correct for multiple testing when using the same data set in the correlation tests and when using species and resin collection as explanatory variables, we adjusted p-values using the Holm method (function: p.adjust, stats package).A significance level (α) of 0.05 was used for all models.The models' original p-values are presented in the results and marked in bold if they were significant after adjustment.

Relationship between resin collection and visited plant species and pollen amino acid profiles
To analyze if resin collection correlated with specialization levels for pollen sources, we conducted a network analysis and calculated H 2 ′ and d′ specialization indices for low and high resin collectors.
As above, a null-model approach was implemented.We additionally compared the proportion of herbs, shrubs, trees, and resinproducing plants visited for pollen collection (Table 2).
We also tested for differences in the concentration of pollen amino acids (Table 2) and the proportional pollen amino acid profiles between the two groups.As above, we conducted a random forest analysis using resin collection as the classification predictor and examined which amino acids were most important for the classification (details in Appendix S1: Statistical analysis).A multidimensional scaling graph, using 1 -Random Forest proximities as distances, was created for data visualization.

| Differences in foraging patterns, resource intake, and nectar collection
The seven stingless bee species differed in their foraging activity.
The seven species also differed in their percentages of returning foragers with no load, nectar, pollen, resin, pollen-nectar, resinnectar, and total pollen (Figure 4, Table 3), with no differences between colonies of the same species (Figure S2).The main differences between species were in the percentage of resin foragers (Table S4).For instance, up to 47% and 42% of T. angustula and T.
ziegleri, respectively, and up to 25% of P. occidentalis foragers collected only resin, while only up to 1% of Scaptotrigona sp. 2, 4% of N.
Results for foragers/minute with each resource were similar to the results for percentages (Table 3, Figure S3, Tables S3 and S4).For

F I G U R E 4
Mean percentage of returning foragers with no load, only nectar, only pollen, only resin, pollen and nectar, resin and nectar, and resin and pollen of the seven different stingless bee species observed in the forest of the reserves.Note the differences between the low resin collectors (N.tristella, Plebeia sp., Scaptotrigona sp. 1, Scaptotrigona sp. 2) and high resin collectors (P.occidentalis, T. ziegleri, and T. angustula) in the percentage of foragers coming back with resin.
example, P. occidentalis, T. ziegleri, and T. angustula invested around five times more workforce into collecting resin than the other species.
Interestingly and contrary to the percentage of only pollen foragers, all bee species invested a similar workforce in collecting only pollen (Table 3, Figure S3, Table S4).However, the overall pollen intake/minute was three to four times higher in Scaptotrigona sp. 1 than in T. angustula and P. occidentalis, or T. ziegleri, respectively (Figure S3).
All bee species collected pollen with similar amino acid profiles TA B L E 3 Differences between species and high and low resin collectors in the proportion of foragers and foragers per minute (calculated with the normalized foraging activity) with each resource.
The total concentration of amino acids in pollen collected did not differ significantly between species (lm: F 6,19 = 1.3, p = .31,Figure 5c).

| Relationship between resin collection and general foraging patterns, resource intake, and nectar collection
High resin collectors tended to be two times more active (higher flight activity) than low resin collectors (lmm: F 1,2.8 = 13.6,p = .04, Figure 3).Moreover, high resin collectors invested 15 and 180 times more into collecting only resin and resin and nectar, respectively, than low resin collectors (Table 3, Figure S3).Also, the high resin collectors tended to invest about four times more workers (per minute) into coming back with some resources, as well as three and two times fewer workers in collecting only nectar and in overall pollen collection, respectively (Table 3, Figure S3).The number of resin foragers/minute did not correlate with the number of pollen or nectar foragers/minute for any species (Table S6).The amount of sucrose collected, and sucrose intake/minute were similar for high and low resin collectors (glmmTMB: χ 2 = 0.007, p = .93;χ 2 = 0.18, p = .7,respectively, Figure 5a,b).
The total concentration of amino acids in pollen was also similar (lmm: F 1,4.9 = 1.5, p = .28,Figure 5c).However, pollen amino acid profiles largely, but not fully overlapped between the two groups (OOB error rate estimate: 42.3%; low resin collectors class error: 0.46; high resin collectors class error: 0.39; Figure 8, Figure S4).The amino acids mainly responsible for the partial separation of the two groups were glutamic acid, proline, alanine, isoleucine, and histidine (Figure S5, Table S7).

| DISCUSS ION
We examined variation in foraging patterns and resource nutritional composition of seven stingless bee species (11 wild colonies) in north-western Ecuador based on field observations, DNA metabarcoding, and chemical analysis of pollen samples.As expected, the number of returning foragers per minute varied among species, as also observed by Leonhardt et al. (2007).Moreover, the species classified as high resin collectors based on their resin intake and the presence of resin compounds in their cuticular profiles tended to have more flights (per minute) than low resin collectors.This increase in activity could be a strategy to compensate for their comparatively lower proportion of food foragers, as they might have to trade-off collecting resin and collecting food resources.Note that we occasionally observed foragers returning with both pollen and resin, which were most likely pollen foragers with minute resin residues from previous foraging trips or as part of their surface profile (Leonhardt et al., 2009), as pollen mixed with resin is unusable as food (Armbruster, 1984).In fact, the observed increase in activity rendered the workforce invested only in pollen collection similar across all species and between high and low resin collectors.Consequently, high resin collectors also tended to invest more workforce into collecting resources overall (note that the surface of the bees' hindlegs often appears sticky due to their unique surface profile and can be mistaken for a resin load, which might have led to slightly underestimating the number of foragers with actually no load).
The percentage of nectar foragers and sucrose amount in nectar also differed between some species, which is contrary to observations on Sumatran and Australian stingless bees (Inoue et al., 1985;Leonhardt et al., 2014).Our study species and colonies also differed in collected sucrose amounts, with the Ptilotrigona occidentalis colony collecting nectar with the highest sucrose content.Sucrose amounts observed ranged from 0.12 to 2.4 mg (percentage 14-72% sucrose), which is similar to reports for other stingless bee species (Inoue et al., 1985;Leonhardt et al., 2007;Roubik & Buchmann, 1984).We additionally observed differences between species in their sucrose intake per minute, with the Ptilotrigona occidentalis colony also showing the highest sucrose intake.Differences in sucrose intake have also been reported among Australian stingless bees (Leonhardt et al., 2014).Contrary to our expectations, sucrose intake was not higher in stingless bees with a comparatively higher foraging activity as observed for Tetragonula carbonaria in Australia (Leonhardt et al., 2014).
The observed differences in sucrose collection might be related to body size and/or honey storage.For example, P. occidentalis collected double the amount of sucrose per minute than the other species.This species has a comparatively large body size with a head width of 3 mm (Lichtenberg et al., 2017) and may therefore require more sucrose than smaller species.The genus Ptilotrigona stores generally less honey than other species (Camargo & Pedro, 2004) and might thus need to compensate for this by collecting higher amounts of sucrose.However, low honey storage has also been reported for the genus Nannotrigona (Rasmussen & Gonzalez, 2017), but our N. tristella colony did not show a higher sucrose intake compared to the other colonies, suggesting that additional factors might explain the observed difference in sucrose intake.
Also contrary to our initial expectations, we did not find significant differences in the amount nor sucrose intake per minute between high and low resin collectors.This suggests that an increased resin intake might not necessarily influence carbohydrate intake.
Note that we included all returning foragers that came back with nectar in these analyses.However, stingless bees often leave their nest with some nectar in their crops (typically less than 1 μL, Inoue et al., 1985;Leonhardt et al., 2007).As we did not check for amounts of nectar in departing foragers, we cannot entirely rule out having included a few foragers returning without an actual load and thus slightly overestimating the number of nectar foragers.
As previously reported and expected, we found that our studied stingless bee species collected pollen from various plant species, but that each species collected most of its pollen from only three plant families as already reported by Vossler (2018) and Garcia nor by pollen protein content and amino acid profile.Instead, different species mostly overlapped in their pollen amino acid profiles which has also been observed in bumblebees (Kriesell et al., 2017), suggesting that they share similar requirements.However, we often had only few pollen samples per species and a limited number of colonies per species.Moreover, we did not check for the foraging range of our studied species or the plant species which were flowering at the time of the study.We therefore refrain from drawing any conclusions on species-specific foraging choices or preferences.
Interestingly, high and low resin collectors foraged pollen from slightly different spectra of plants, which was independent of the plants' life forms or whether the plants additionally produced resin, suggesting that the bees do not preferentially collect pollen from plants that additionally produce resin.Moreover, amino acid profiles of pollen collected by the two groups tended to differ, indicating that a high resin intake may affect the spectrum of plants visited for pollen foraging.However, the high resin collectors also show a solitary foraging strategy, while, for example, species of the genus Scaptotrigona show forager recruitment (Table 1).Some Nannotrigona and Plebeia species are considered solitary foragers (Aguilar et al., 2005;Lichtenberg et al., 2010;Slaa et al., 1997).
However, they have also been observed recruiting nestmates to food sources, and Plebeia tica appears to be a more effective recruiter than T. angustula (Aguilar et al., 2005).These differences in recruitment strategies may also explain the differences in visited plant and pollen amino acid profiles as observed in our study.Group foragers and mass recruiter species tend to dominate food patches, often driving off solitary foragers, which then visit other plants or access resources before the dominant species arrive (Cairns et al., 2005;Nagamitsu & Inoue, 1997).Future research should ideally compare recruiting species differing in resin intake to disentangle effects of foraging strategies and resin.
It is noteworthy that species classified here as high resin collectors (P.occidentalis and T. angustula) rely more heavily on resin for defense (see Table 1) compared to low resin collectors.For instance, the high resin collector T. angustula applies resin to predators and places resin droplets around the nest entry to trap intruders (Wittmann, 1985, C. Rasmussen pers. obs.).In contrast, the low resin collector N. tristella tends to hide in it is nest and seals the nest entry with wax (Rasmussen & Gonzalez, 2017, G. N. Villagómez pers. obs.).
Species within the Scaptotrigona genus exhibit strong physical aggression when threatened, such as attacking and biting intruders (e.g., Couvillon et al., 2008;Jungnickel et al., 2004;G. N. Villagómez pers. obs.).These differences in defense strategies may partially explain the significant effort invested in resin collection by the high resin collectors.
Additional limitations of our study are the limited number of observed colonies per species and that all observations were conducted in a single season.Environmental and colony-specific factors, such as predator pressure, a colony's developmental or feeding stage, time of day, and season, affect resource collection and resource intake by colonies, including resin collection (Hilário et al., 2000(Hilário et al., , 2012;;Hofstede & Sommeijer, 2006;Leonhardt & Blüthgen, 2009;Newis et al., 2023;Nunes-Silva et al., 2010;Shanahan & Spivak, 2021).To fully elucidate the effect of resin collection on stingless bee foraging and resource intake, repeated observations across different times of the year and including additional colonies as well as controlled experiments that manipulate resin and pollen storage within nests (e.g., Newis et al., 2023) are needed.
To conclude, our findings highlight the importance of studying the foraging ecology and resource intake of different stingless bee species in their natural environment to understand how differences in life-history traits may affect species' foraging patterns.Such differences may be innate or a consequence of specific colony states (e.g., colony reserves, developmental stage) (Biesmeijer & Slaa, 2004).As we have only a few colonies for some species, we cannot disentangle these two factors.We do show, however, that the collection of a non-food resource, such as resin, appears to affect stingless bee foraging patterns, plant choices, and resource intake.Plant resins are an ideal material for construction and defense (Armbruster, 1984;Chui et al., 2022;Greco et al., 2010;Roubik, 2006), and can even be incorporated into their surface profile as protection against predation and microbes (Leonhardt, 2017).They have therefore become a vital resource for many stingless bee species.Their importance could have led to species modifying their foraging behavior, that is, increasing
et al. (2021), and in the Appendix S1.Sequencing reads were quality filtered, denoised to amplicon sequence variants (ASVs), chimera filtered, and taxonomically classified with VSEARCH(Rognes et al., 2016) against custom reference databases for the study region created with the BCdatabaser(Keller et al., 2020).Read counts per sample were transformed to relative read abundances (RRAs) by dividing the number of reads per taxon and samples by the sample sum.We excluded samples with <100 reads and the taxa accounting for <1% of reads per sample to determine the most abundant plant species visited (phyloseq R-package,McMurdie & Holmes, 2013) (details in Appendix S1).

F I G U R E 5
Sucrose amount in nectar collected (a), sucrose intake (mg) per minute (calculated based on the normalized flight activity) (b), and total pollen amino acid (μg/mg pollen) collected (c) by seven different stingless bee species observed in the forest of the reserves.Species are grouped into low (white boxplots) and high (gray boxplots) resin collectors.Box plots display the median (thick bar), lower and upper quartile (boxes), and minimum and maximum values (whiskers) of the data set.Black diamonds represent the mean value of the data set.In the case of sucrose amount and intake, N. tristella is represented with 31 nectar samples (n), Plebeia sp.n = 22, P. occidentalis n = 54, Scaptotrigona sp. 1 n = 69, Scaptotrigona sp. 2 n = 23, T. ziegleri n = 55, and T. angustula n = 58.In the case of pollen amino acids n = 3, except for N. tristella with n = 2, Scaptotrigona sp. 1 with n = 5, and T. angustula with n = 7.

F
Pollen collection networks.Bipartite network for seven stingless bee species (top) and proportion of plant species (bottom) visited for pollen collection (a).Bipartite network showing stingless bee species pooled according to their resin collection preference (top) and proportion of plant species (bottom) visited for pollen collection (b).The bottom block width represents the overall proportions of a given plant species across stingless bee species pollen loads (Plant taxa accounting for more than 10% of reads are shown).Nannotrigona tristella is represented with 12 pollen samples, Plebeia sp. with 3, P. occidentalis with 9, Scaptotrigona sp. 1 with 29, Scaptotrigona sp. 2 with 20, T. ziegleri with 9, and T. angustula with 18.

F I G U R E 7
Proportions of plant life forms (a) and resin-producing plants (b) (both mean percentages) within the spectrum of plants visited for pollen collection by the seven different stingless bee species observed in the forest of the reserves.Data comprise 12 pollen loads from N. tristella (low resin collector -lrc-), 3 from Plebeia sp.(lrc), 9 from P. occidentalis (high resin collector -hrc-), 29 from Scaptotrigona sp. 1 (lrc), 20 from Scaptotrigona sp. 2 (lrc), 9 from T. ziegleri (hrc), and 18 T. angustula (hrc).BulleBueno et al. (2023).Piperaceae, Clusiaceae, Moraceae, and Cannabaceae were the most commonly visited families.Also, some species (i.e., T. ziegleri, Plebeia sp., and Scaptotrigona sp. 2) collected pollen from fewer plants, indicating that they might prefer pollen from particular plants.Contrary to our expectations, the differences between the visited plants were not driven by the plants' life form

F
Multidimensional scaling based on Random Forest proximities, showing similarities in pollen amino acid profiles collected by the seven different stingless bee species in this study.Species are grouped into low (circles) and high (triangles) resin collectors.Different colors indicate different stingless bee species, and each dot represents one pooled pollen sample per colony and day.Except for N. tristella with n = 2, Scaptotrigona sp. 1 with n = 5 (2-3 from each colony), and T. angustula with n = 7 (2-3 from each colony), all other species with n = 3.
Implemented statistical models for analyzing species differences and differences between resin collection categories (explanatory variables) in the studied variables (response variables).
we assessed if colony ID and (or) nest location (i.e., reserve) improved the explanatory force of our models and should therefore be included as random factors (details in Appendix S1: Statistical analysis).Models without random factor(s) had smaller AIC values (Table